The overall aim of this proposal is to better understand the cellular and molecular underpinnings of hematopoietic ontogeny. Our studies during the first three cycles of this grant have focused on the initial emergence of hematopoietic potential in the mammalian embryo and the fate of that potential within the conceptus. We have defined the appearance of two overlapping waves of hematopoietic potential in the yolk sac prior to the emergence of hematopoietic stem cells. The first primitive wave primarily generates red cells to establish a functional circulation. The second wave consists of definitive erythroid, megakaryocyte, and multiple myeloid progenitors, recently termed erythro-myeloid progenitors (EMP). In preliminary studies we have determined that EMP display a unique cell surface phenotype and depend on Runx1 for their normal emergence. In this fourth grant cycle, we will elucidate the temporal and spatial emergence of EMP from hemogenic endothelium in the mouse embryo, and more fully define their hematopoietic lineage potential using in vitro in and vivo assays. Furthermore, we will investigate the role of blood flow and of Runx1 dosage on EMP emergence. Finally, we will test the hypothesis that definitive hematopoiesis in differentiating embryonic stem cells mimics EMP emergence in the yolk sac. A better understanding of the early ontogeny of the hematopoiesis in mammalian embryos, and the parallel processes that occur in differentiating embryonic stem cells, will ultimately lead to achieving our long-term goal of generating hematopoietic progenitor and stem cells for use in cell replacement therapies. PUBLIC HEALTH RELEVANCE: The first blood cells in the embryo arise in two waves in the yolk sac before blood cell production moves to the liver of the fetus. The goal of this research is to better understand the emergence of these first blood cell progenitors in embryos and also in cultured embryonic stem cells. Our ultimate goal is to produce blood cells from embryonic stem cells for use in cell replacement therapies.